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Background: Colorectal cancer (CRC) is one of the most common malignancies worldwide. Given its insidious onset, the condition often already progresses to advanced stage when symptoms occur. Thus, early diagnosis is of great significance for timely clinical intervention, efficacy enhancement, and prognostic improvement. Featuring high throughput, fastness, and rich information, next generation sequencing (NGS) can greatly shorten the detection time, which is a widely used detection technique at present., Aim: To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC, and to establish a technological platform for CRC screening, diagnosis, and efficacy monitoring through fecal DNA detection., Methods: NGS was used to sequence the stool DNA of patients with CRC, which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease, as well as the tumor tissues of CRC patients. Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened, and their significances in diagnosing CRC and predicting patients' prognosis were comprehensively evaluated., Results: High mutation frequencies of TP53 , APC, and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery. Contrastively, no pathogenic mutations of the above three genes were noted in the postoperative stools, the normal controls, or the benign intestinal disease group. This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients. The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent. Compared to the postoperative stools and the stools in the two control groups, the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools ( χ 2 = 7.328, P < 0.05; χ 2 = 4.219, P < 0.05), suggesting that fecal TP53 and KRAS genes can be used for CRC screening, diagnosis, and prognostic prediction. No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups ( χ 2 = 0.878, P > 0.05), so further analysis with larger sample size is required. Among CRC patients, the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools, with a true positive rate of 59.26%, while the pathogenic mutation sites of KRAS occurred in 10 stools, with a true positive rate of 37.04%. The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67% (18/27) and 68.97%, respectively, both of which were higher than those of TP53 or KRAS mutation detection alone, suggesting that the combined genetic testing can improve the CRC detection rate. The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T (mutation start and stop positions were both 7579436 for the former, while 55249164 for the latter) were found in the preoperative stools and tumor tissues. These "undetected" mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression, which needs to be confirmed through further research. Some mutations of "unknown clinical significance" were found in such genes as TP53 , PTEN , KRAS , BRAF , AKT1 , and PIK3CA , whose clinical values is worthy of further exploration., Conclusion: NGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis. Fecal TP53 and KRAS can be used as specific genes for the screening, diagnosis, prognostic prediction, and recurrence monitoring of CRC. Moreover, the combined testing of TP53 and KRAS genes can improve the CRC detection rate., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)